We are pursuing investigations of metacyclogenesis, or maturation to the virulent promastigote stage of the parasite's life cycle, using a genetic complementation strategy to correct the defect in maturation mutants, in collaboration with David Sacks at NIH and Steven Beverley at Harvard. A genomic cosmid library made from virulent parasite DNA is transfected into avirulent parasite mutant lines from the same strain. Cosmids that 'rescue' virulence can be recovered by screening for the virulent phenotype. As part of this project, we discovered a morphological change in agar cultures as the colonies mature and undergo metacyclogenesis, which allows selection of a mutant phenotype. Currently we are working thru the transfection and selection of the mutant clones that recover the ability to differentiate. Several rescued clones have been isolated and are being re-transfected into the mutant to confirm the change in phenotype. The next step is to map the cosmids, then isolate and sequence the gene(s) responsible for the phenotypic change. These strategies should allow an unbiased isolation of genes relevant to parasite differentiation and virulence, both at the structural and regulatory levels. Identification of these 'virulent' genes will allow the construction of knockout vectors that will be used to make attenuated strains of the parasite to be evaluated as vaccines.